There has been much interest in recent years in antibodies and their fragments. It is well known that complete antibody molecules comprise four polypeptide chains, two heavy chains and two light chains. Each light chain consists of two domains, the N-terminal domain being known as the variable or VL domain and the C-terminal domain being known as the constant or CL domain. Each heavy chain consists of four or five domains, depending on the class of the antibody. The N-terminal domain is known as the variable or VH domain. The next domain is known as the first constant or CH1 domain. The next part of each heavy chain is known as the hinge region and this is then followed by the second, third and, in some cases, fourth constant or CH2, CH3 and CH4 domains respectively.
In an assembled antibody, the VL and VH domains associate together to form an antigen binding site. Also the CL and CH1 domains associate together to keep one heavy chain associated with one light chain. The two heavy-light chain heterodimers associate together partly by interaction of the CH2, CH3 and CH4 domains of the two heavy chains and partly because of interaction between the hinge regions on the two heavy chains.
Each heavy chain hinge region includes at least one, and often several, cysteine residues. In the assembled antibody, the cysteine residues in the heavy chains are aligned so that disulphide bonds can be formed between the cysteine residues in the hinge regions covalently bonding the two heavy-light chain heterodimers together. Thus, fully assembled antibodies are bivalent in that they have two antigen binding sites.
It has been known for some long time that if the disulphide bonds in an antibody's hinge region are broken by mild reduction, it is possible to produce a monovalent antibody comprising a single heavy-light chain heterodimer.
It has also been known for some long time that treatment of antibodies with certain proteolytic enzymes leads to the production of various antibody fragments. For instance, if an antibody is cleaved close to the N-terminal side of each hinge region, two antibody binding fragments (Fab) and one constant region fragment (Fc) are produced. Each Fab fragment comprises the light chain associated with a truncated heavy chain comprising only the VH and CH1 domains. The Fc portion comprises the remaining domains of the heavy chains held together by the hinge region.
Alternatively, the antibody may be cleaved close to the C-terminal side of the hinge. This produces a fragment known as the F(ab ).sub.2 fragment. This essentially comprises two Fab fragments but with the CH1 domains still attached to the hinge regions. Thus, the F(ab ).sub.2 fragment is a bivalent fragment having the two antigen binding sites linked together by the hinge region.
It has also proved to be possible, by careful control of digestion conditions, to cleave an antibody between the VL and CL and between the VH and CH1 domains. This gives rise to two fragments known as FV fragments. Each Fv fragment comprises a VL and a VE domain associated with one another. Each Fv fragment is monovalent for antigen binding.
In more recent years, there has been much interest in producing antibodies or their fragments by use of recombinant DNA technology. The patent literature is replete with disclosures in this area. Recombinant DNA technology has been used not only to reproduce natural antibodies but also to produce novel antibodies. For instance, it is now possible to produce chimeric antibodies, wherein the variable domains from one species are linked to constant domains from another species.
It is also possible to produce CDR-grafted antibodies, in which the regions in the VH and VL domains responsible for antigen binding activity (usually referred to as the Complementarity Determining Regions) have been changed in sequence so that a different antigen can be bound. CDR-grafting an antibody is a useful procedure in that it allows a specificity from, for instance, a mouse monoclonal antibody to be transferred to a human antibody without altering the essentially human nature of the antibody. This has advantages where it is desired to use the antibody in vivo.
Relatively recently, there has been interest in producing Fv fragments by recombinant DNA technology. For instance, production of Fv fragments by recombinant DNA technology has been suggested by Skerra and Pluckthun 1!. (A list of references is appended at the end of the description.) The production of Fv-producing recombinant host cells has been described in published International patent application Ser. Nos. WO 89/02465 and WO 89/09825.
It is envisaged that Fv fragments will be of particular use in in vivo diagnosis and in therapy because of their small size as compared to Fab fragments or complete antibodies. This will mean that they are likely to be less antigenic and more able to penetrate tissue. In particular, it is envisaged that they may be able to penetrate cancerous tissue and therefore deliver a cytotoxic drug or diagnostic label to the site of the cancer.
Fv fragments have the disadvantage that they are monovalent with respect to antigen binding. Most naturally occurring antigens are multivalent, i.e. they display multiple antigenic determinants. Therefore, the Fv fragments, with only one antigen binding site per molecule, will bind to such antigens less strongly than the bivalent whole antibody or an F(ab ).sub.2 fragment. This may reduce the usefulness of the Fv fragment, for example in targeting a cell surface antigen.